Characterization of the Mechanistic Linkages Between Iodothyronine Deiodinase Inhibition and Impaired Thyroid-Mediated Growth and Development in Xenopus laevis Using Iopanoic Acid.

Iodothyronine deiodinases (DIO) are key enzymes that influence tissue-specific thyroid hormone levels during thyroid-mediated amphibian metamorphosis. Within the larger context of evaluating chemicals for thyroid system disrupting potential, chemical activity toward DIOs is being evaluated using high-throughput in vitro screening assays as part of U.S.

Targeted Pathway-based In Vivo Testing Using Thyroperoxidase Inhibition to Evaluate Plasma Thyroxine as a Surrogate Metric of Metamorphic Success in Model Amphibian Xenopus laevis.

Chemical safety evaluation is in the midst of a transition from traditional whole-animal toxicity testing to molecular pathway-based in vitro assays and in silico modeling. However, to facilitate the shift in reliance on apical effects for risk assessment to predictive surrogate metrics having characterized linkages to chemical mechanisms of action, targeted in vivo testing is necessary to establish these predictive relationships. In this study, we demonstrate a means to predict thyroid-related metamorphic success in the model amphibian Xenopus laevis using relevant biochemical measurements during early prometamorphosis.

Evaluating Chemicals for Thyroid Disruption: Opportunities and Challenges with in Vitro Testing and Adverse Outcome Pathway Approaches.

Background: Extensive clinical and experimental research documents the potential for chemical disruption of thyroid hormone (TH) signaling through multiple molecular targets. Perturbation of TH signaling can lead to abnormal brain development, cognitive impairments, and other adverse outcomes in humans and wildlife. To increase chemical safety screening efficiency and reduce vertebrate animal testing, in vitro assays that identify chemical interactions with molecular targets of the thyroid system have been developed and implemented.

Linking mechanistic toxicology to population models in forecasting recovery from chemical stress: A case study from Jackfish Bay, Ontario, Canada.

Recovery of fish and wildlife populations after stressor mitigation serves as a basis for evaluating remediation success. Unfortunately, effectively monitoring population status on a routine basis can be difficult and costly. In the present study, the authors describe a framework that can be applied in conjunction with field monitoring efforts (e.

Molecular target sequence similarity as a basis for species extrapolation to assess the ecological risk of chemicals with known modes of action.

It is not feasible to conduct toxicity tests with all species that may be impacted by chemical exposures. Therefore, cross-species extrapolation is fundamental to environmental risk assessment. Recognition of the impracticality of generating empirical, whole organism, toxicity data for the extensive universe of chemicals in commerce has been an impetus driving the field of predictive toxicology.

Current perspectives on the use of alternative species in human health and ecological hazard assessments.

Background: Traditional animal toxicity tests can be time and resource intensive, thereby limiting the number of chemicals that can be comprehensively tested for potential hazards to humans and/or to the environment.

Objective: We compared several types of data to demonstrate how alternative models can be used to inform both human and ecological risk assessment.

Methods: We reviewed and compared data derived from high throughput in vitro assays to fish reproductive tests for seven chemicals.

Assessment of status of white sucker (Catostomus commersoni) populations exposed to bleached kraft pulp mill effluent.

Credible ecological risk assessments often need to include analysis of population-level impacts. In the present study, a predictive model was developed to investigate population dynamics for white sucker (Catostomus commersoni) exposed to pulp mill effluent at a well-studied site in Jackfish Bay, Lake Superior, Canada. The model uniquely combines a Leslie population projection matrix and the logistic equation to translate changes in the fecundity and the age structure of a breeding population of white sucker exposed to pulp mill effluent to alterations in population growth rate.

Inhibition of the thyroid hormone pathway in Xenopus laevis by 2-mercaptobenzothiazole.

Determining the effects of chemicals on the thyroid system is an important aspect of evaluating chemical safety from an endocrine disrupter perspective. Since there are numerous chemicals to test and limited resources, prioritizing chemicals for subsequent in vivo testing is critical. 2-Mercaptobenzothiazole (MBT), a high production volume chemical, was tested and shown to inhibit thyroid peroxidase (TPO) enzyme activity in vitro, a key enzyme necessary for the synthesis of thyroid hormone.

Control of pituitary thyroid-stimulating hormone synthesis and secretion by thyroid hormones during Xenopus metamorphosis.

We used ex vivo and in vivo experiments with Xenopus laevis tadpoles to examine the hypothesis that the set-point for negative feedback on pituitary thyroid-stimulating hormone (TSH) synthesis and secretion by thyroid hormones (THs) increases as metamorphosis progresses to allow for the previously documented concomitant increase in serum TH concentrations and pituitary TSH mRNA expression during this transformative process. First, pituitaries from climactic tadpoles were cultured for up to 96 h to characterize the ability of pituitary explants to synthesize and secrete TSHβ in the absence of hypothalamic and circulating hormones. Next, pituitary explants from tadpoles NF stages 54-66 were exposed to physiologically-relevant concentrations of THs to determine whether stage-specific differences exist in pituitary sensitivity to negative feedback by THs.

Thyroid-stimulating hormone (TSH): measurement of intracellular, secreted, and circulating hormone in Xenopus laevis and Xenopus tropicalis.

Thyroid-stimulating hormone (TSH) is an important regulator of the hypothalamic-pituitary-thyroid (HPT) axis in Xenopus laevis. To evaluate the role of this hormone on developing tadpoles, immunologically-based Western blots and sandwich ELISAs were developed for measuring intracellular (within pituitaries), secreted (ex vivo pituitary culture), and circulating (serum) amounts. Despite the small size of the tadpoles, these methods were able to easily measure intracellular and secreted TSH, and circulating TSH was measurable in situations where high levels were induced.

Adverse outcome pathways: a conceptual framework to support ecotoxicology research and risk assessment.

Ecological risk assessors face increasing demands to assess more chemicals, with greater speed and accuracy, and to do so using fewer resources and experimental animals. New approaches in biological and computational sciences may be able to generate mechanistic information that could help in meeting these challenges. However, to use mechanistic data to support chemical assessments, there is a need for effective translation of this information into endpoints meaningful to ecological risk-effects on survival, development, and reproduction in individual organisms and, by extension, impacts on populations.

Inhibition of thyroid hormone release from cultured amphibian thyroid glands by methimazole, 6-propylthiouracil, and perchlorate.

Thyroid gland explant cultures from prometamorphic Xenopus laevis tadpoles were evaluated for their utility in assessing chemicals for thyroid hormone (TH) synthesis disruption. The response of cultured thyroid glands to bovine thyroid stimulating hormone (bTSH) and the TH synthesis inhibitors methimazole, 6-propylthiouracil, and perchlorate was determined. Thyroid glands continuously exposed for 12 days to graded concentrations of bTSH released thyroxine (T4) in a dose-dependent manner.

In vivo assessment and potential diagnosis of xenobiotics that perturb the thyroid pathway: Proteomic analysis of Xenopus laevis brain tissue following exposure to model T4 inhibitors.

As part of a multi-endpoint systems approach to develop comprehensive methods for assessing endocrine stressors in vertebrates, differential protein profiling was used to investigate expression patterns in the brain of the amphibian model (Xenopus laevis) following in vivo exposure to a suite of T4 synthesis inhibitors. We specifically address the application of Two Dimensional Polyacrylamide Gel Electrophoresis (2D PAGE), Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) and LC-MS/MS to assess changes in relative protein expression levels. 2D PAGE and iTRAQ proved to be effective complementary techniques for distinguishing protein changes in the developing amphibian brain in response to T4 synthesis inhibition.

Early temporal effects of three thyroid hormone synthesis inhibitors in Xenopus laevis.

Thyroid axis disruption is an important consideration when evaluating risks associated with chemicals. Bioassay methods that include thyroid-related endpoints have been developed in a variety of species, including amphibians, whose metamorphic development is thyroid hormone (TH)-dependent. Inhibition of TH synthesis in these species leads to developmental delay, and assays designed to capture these effects take several weeks to complete.

Progress towards development of an amphibian-based thyroid screening assay using Xenopus laevis. Organismal and thyroidal responses to the model compounds 6-propylthiouracil, methimazole, and thyroxine.

In response to the initial Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) recommendations, research was conducted on the development of a Xenopus laevis based tail resorption assay for evaluating thyroid axis disruption. This research highlighted key limitations associated with relying on tail resorption as a measure of anti/thyroid activity. The most critical limitation being that tail tissues of tadpoles at metamorphic climax are insensitive to perturbation by thyroid axis agonists/antagonists.

Metamorphic inhibition of Xenopus laevis by sodium perchlorate: effects on development and thyroid histology.

The perchlorate anion inhibits thyroid hormone (TH) synthesis via inhibition of the sodium-iodide symporter. It is, therefore, a good model chemical to aid in the development of a bioassay to screen chemicals for affects on thyroid function. Xenopus laevis larvae were exposed to sodium perchlorate during metamorphosis, a period of TH-dependent development, in two experiments.

Assessment of environmental stressors potentially responsible for malformations in North American anuran amphibians.

Several species of anuran amphibians from different regions across North America have recently exhibited an increased occurrence of malformations, predominantly of the hindlimb. Research concerning the potential causes of these malformations has focused extensively on three stressors: chemical contaminants, ultraviolet (UV) radiation, and parasitic trematodes. In this overview of recent work with each of these stressors, we assess their plausibility as contributors to the malformations observed in field-collected amphibians.

Comparing the effects of stage and duration of retinoic Acid exposure on amphibian limb development: chronic exposure results in mortality, not limb malformations.

Recently, high frequencies of malformations have been reported in amphibians across the United States. It has been suggested that the malformations may be the result of xenobiotic disruption of retinoid signaling pathways during embryogenesis and tadpole development. Therefore, a series of experiments were undertaken to examine life-stage specific effects of continuous retinoid exposure on Xenopus laevis.

Developmental toxicity of methoprene and several degradation products in Xenopus laevis.

Methoprene is an insect juvenile growth hormone mimic, which inhibits pupation and is used for the control of emergent insect pests such as mosquitoes. Researchers have hypothesized that methoprene use in US may be a contributing factor to the recent increase in malformed amphibians. However, little is known concerning the developmental toxicity of methoprene and its degradation products in amphibians.

Xenoendocrine disrupters-tiered screening and testing: filling key data gaps.

The US Environmental Protection Agency (EPA) is developing a screening and testing program for endocrine disrupting chemicals (EDCs) to detect alterations of hypothalamic-pituitary-gonadal (HPG) function, estrogen (ER), androgen (AR) and thyroid hormone synthesis and AR and ER receptor-mediated effects in mammals and other animals. High priority chemicals would be evaluated in the Tier 1 Screening (T1S) battery and chemicals positive in T1S would then be tested (Tier 2). T1S includes in vitro ER and AR receptor binding and/or gene expression, an assessment of steroidogenesis and mammalian (rat) and nonmammalian in vivo assays (Table 1).

Assessment of the risk of solar ultraviolet radiation to amphibians. III. Prediction of impacts in selected northern midwestern wetlands.

Solar ultraviolet radiation, especially UVB (280-320 nm), has been hypothesized to be at least partially responsible for adverse effects (e.g., declines and malformations) in amphibian species throughout the world.

Assessment of the risk of solar ultraviolet radiation to amphibians. I. Dose-dependent induction of hindlimb malformations in the northern leopard frog (Rana pipiens).

A number of environmental stressors have been hypothesized as responsible for recent increases in limb malformations in several species of North American amphibians. The purpose of this study was to generate dose-response data suitable for assessing the potential role of solar ultraviolet (UV) radiation in causing limb malformations in a species in which this phenomenon seemingly is particularly prevalent, the northern leopard frog (Rana pipiens). Frogs were exposed from early embryonic stages through complete metamorphosis to varying natural sunlight regimes, including unaltered (100%) sunlight, sunlight subjected to neutral density filtration to achieve relative intensities of 85%, 75%, 65%, 50%, and 25% of unaltered sunlight, and sunlight filtered with glass or acrylamide to attenuate, respectively, the UVB (290-320 nm) and UVB plus UVA (290-380 nm) portions of the spectrum.

Iodine species and the endocrine system: thyroid hormone levels in adult Danio rerio and developing Xenopus laevis.

Recently a new approach for the analysis of iodinated organic species in human serum has been developed using liquid chromatography-inductively coupled plasma-mass spectrometry (LC-ICP-MS). This method enables quantification of iodide, T4 and T3, as well as reverse T3 (rT3) and the synthetic precursors of TH, monoiodotyrosine (MIT), and diiodotyrosine (DIT) in a single injection. In this work, the LC-ICP-MS approach was used to analyze whole-body homogenates of adult male and female zebrafish (Danio rerio) and tadpoles of the African clawed frog (Xenopus laevis) at two different developmental stages (NF58 and 61) according to Nieuwkoop and Faber.

Ambient solar UV radiation causes mortality in larvae of three species of Rana under controlled exposure conditions.

Recent reports concerning the lethal effects of solar ultraviolet-B (UV-B) (290-320 nm) radiation on amphibians suggest that this stressor has the potential to impact some amphibian populations. In this study embryos and larvae of three anuran species, Rana pipiens, Rana clamitans and Rana septentrionalis, were exposed to full-spectrum solar radiation and solar radiation filtered to attenuate UV-B radiation or UV-B and ultraviolet-A (UV-A) (290-380 nm) radiation to determine the effects of each wavelength range on embryo and larval survival. Ambient levels of solar radiation were found to be lethal to all three species under exposure conditions that eliminated shade and refuge.

Effects of the mammalian antiandrogen vinclozolin on development and reproduction of the fathead minnow (Pimephales promelas).

Previous work with the chlorinated fungicide vinclozolin and its metabolites, 2-{[(3,5-dichlorophenyl)-carbamoyl]oxy}-2-methyl-3-butenoic acid (M1) and 3',5'-dichloro-2-hydroxy-2-methylbut-3-enanilide (M2), indicated antiandrogenic properties expressed in vivo as abnormalities in sexual differentiation of male rats after maternal exposures. In this study, we attempted to determine whether vinclozolin might also exhibit antiandrogenic properties in a model fish species, the fathead minnow, Pimephales promelas. In one study, embryonic (<6 h old) fathead minnows were exposed for approximately 34 days to five toxicant concentrations, ranging from 90 to 1200 µg l(-1), delivered via a flow-through diluter.